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Administrative data

Description of key information

Four good quality studies provide the endpoint data for repeated dose toxicity.


A chronic (180d) oral study produced a LOAEL of 30 mg/kg (Yeary, 1969). In support a 28 day study produced a NOAEL of 5 mg/kg (Innospec, 1998).


A 90 day sub chronic inhalation study produced a LOAEC of 3 mg/ m3 (Nikula, 1993). In support a 14 day study obtained a NOAEC of 5.0 mg/m3 (Sun et al, 1991).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Principles of method if other than guideline:
No exact method guidelines given in publication, but a chronic oral study on dogs is described.
GLP compliance:
not specified
Species:
dog
Strain:
not specified
Sex:
not specified
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
dogs were administered ferrocene in gelatin capsules once each day at 30, 100, 300, and 1000 mg/kg. Controls were given empty gelatin capsules
Duration of treatment / exposure:
180 days
Frequency of treatment:
Daily
Control animals:
yes, concurrent no treatment
Sacrifice and pathology:
Animals were sacrificed at 12 and 24 weeks or observed for 1 year or 26 months after the 12 week treatment period.
Clinical signs:
no effects observed
Description (incidence and severity):
there were no deaths
Mortality:
no mortality observed
Description (incidence):
there were no deaths
Key result
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Basis for effect level:
other: Hemosiderosis was observed
Critical effects observed:
not specified

Hemosiderosis was observed at 30, 100, and 300 mg/kg ferrocene at 6 months and 1000 mg/kg at 3 months. There was a dose related accumulation of iron.

There was a reversible decrease in hemoglobin, packed cell volume, and erythrocyte counts with greatest change occurring during the first 4 weeks at doses of 300 mg/kg. Cirrhosis was seen with 1000 and 300 mg/kg ferrocene, and testicular hypoplasia occurred with 300 and 100 mg/kg ferrocene.

Dogs observed for 12 to 26 months after the 6 month treatment period showed no latent effects from massive iron overload. The ferrocene induced hepatic iron overload was reduced by repeated venesection and removal of large quantities of iron. This resulted in mobilization of the storage iron for hemoglobin synthesis.

All other parameters were normal

Executive summary:

Mongrel-dogs were administered ferrocene in gelatin capsules once each day at 30, 100, 300, and 1000 mg/kg. Controls were given empty gelatin capsules.

Animals were sacrificed at 12 and 24 weeks or observed for 1 year or 26 months after the 6 month treatment period.

Liver biopsies were taken from ferrocene treated dogs (10th month) and from dogs used in the ferrous reversibility study. At necropsy, all tissues were examined grossly and later histologically. Blood and urine samples were collected at intervals and measured for hematologic and biochemical parameters including iron absorption and storage. There were no deaths.

Hemosiderosis was observed at 30, 100, and 300 mg/kg ferrocene at 6 months and 1000 mg/kg at 3 months. There was a dose related accumulation of iron. There was a reversible decrease in hemoglobin, packed cell volume, and erythrocyte counts with greatest change occurring during the first 4 weeks at doses of 300 mg/kg. Cirrhosis was seen with 300 and 1000 mg/kg ferrocene, and testicular hypoplasia occurred with 100 and 300 mg/kg ferrocene or 150 and 500 mg/kg ferrous-sulfate at 6 months.

Dogs observed for 12 to 26 months after the 6 month treatment period showed no latent effects from massive iron overload. The ferrocene induced hepatic iron overload was reduced by repeated venesection and removal of large quantities of iron. This resulted in mobilization of the storage iron for hemoglobin synthesis.

All other parameters were normal.


Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Fifty one Sprague-Dawley rats (25 males and 26 females) were obtained from Charles River (U.K.) Limited, Manston, Kent, England on 29 March
1988. They were ordered to be ca 4 weeks old (male - ca 85 g, female - 60 g) on arrival.

Twenty males and 20 females were allocated to treatment groups and allowed to acclimatise for 7 days.

Rats were housed in a barrier maintained room at a room temperature normally of 20°C ± 2°C and a target relative humidity of 55% ± 10% (both automatically controlled), with 15-20 air changes per hour.

A 12 h light/dark cycle was controlled by a time switch, light hours being 0700-1900 h. Rats were housed either 2 or 3 of one sex per cage in suspended polypropylene cages (overall dimensions ca 420 x 270 x 200 mm) with stainless steel wire grid tops and bottoms. Beneath each cage was suspended a polypropylene tray containing absorbent paper. Tray paper was changed as required during the study. Each cage had a polypropylene water bottle (total capacity 300 ml) with rubber washer and melamine cap. Bottles were cleaned on a rota basis

During the course of the study, tap water and S.O.S. Rat and Mouse No.1 Expanded Fine Ground Diet were available to the rats ad libitum. Each day, following completion of all other work, floors were swept then washed with disinfectant solution. Once each week walls, ceiling, benches and racking within the animal room were washed with disinfectant solution
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
Ferrocene was administered orally at varying concentrations via the diet for 4 weeks continual dosing whereupon all animals were killed. Fresh diets were prepared twice each week in S.O.S. Rat and Mouse No. 1 Expanded Fine Ground diet. The method of preparation was by direct admixture of sieved test material to untreated diet and blending for 20 min in a Winkworth change drum mixer.

Each mixed batch was stored in a closed container at ambient temperature.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Chosen dose given in diet
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Clinical signs:
no effects observed
Description (incidence and severity):
There were no premature deaths. There were no clinical signs that could be attributed to dosing with Ferrocene
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths. There were no clinical signs that could be attributed to dosing with Ferrocene
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose males and females showed a reduction in body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
High dose males and females showed a reduction in food consumption
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no visible intergroup differences in either sex.
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
High dose group males and females showed some effects; these were of small magnitude so were considered to be biologically unimportant
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight showed a marked increase in the male High dose group and equivocal increases at the other dose levels. High dose females showed amoderate increase in liver weight (absolute weight and after covariance analysis)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the kidneys, discolouration and a mottled or granular appearance were noted in most male animals receiving Ferrocene.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver, centrilobular hepatocyte hypertrophy (probably a mild non-toxic, adaptive response) was seen in all High dose group animals of both sexes, some Intermediate dose males and females and one Low dose female.
Key result
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Critical effects observed:
not specified
Conclusions:
In conclusion, dosing Sprague-Dawley rats for 4 weeks with Ferrocene via the diet produced effects on male and female RBC parameters (25 and 125 mg/kg/day) leading to haemosiderin deposition in liver (125 mg/kg/day) and spleen (25 and 125 mg/kg/day).

Depressions of body weight gain and food consumption were seen in 125 mg/kg/day dose group of both sexes. In addition 25 and 125 mg/kg/day dose male groups showed intracytoplasmic hyaline droplets in the kidney cortical tubular epithelium. Some 5 mg/kg/day dose males showed similar but minimal changes
Executive summary:

Dosing Sprague-Dawley rats for 4 weeks with up to 125 mg Ferrocene/kg/day produced signs of toxicity in both sexes.

Depressions of body weight gain and food consumption were seen at the 125 mg/kg/day dose level in both sexes. Disturbances of red cell parameters were seen in males and females at the 25 and 125 mg/kg/day dose levels (more marked in males). The findings tended to be more marked in the 125 mg/kg/day dose groups with several animals showing polychromasia. These findings viewed with the histopathological findings of haemosiderin in the liver and its increased deposition in the spleens of the 25 and 125 mg/kg/day dose groups of both sexes indicates an effect of Ferrocene on red blood cells.

Histopathological examination of male kidneys showed hyaline droplet formation in the cortical tubular epithelium of animals receiving Ferrocene. This finding represents an early toxic effect which could progress to tubular degeneration and is a recognised effect of hydrocarbons on the male rat kidney. The gross colour changes seen in some animals at autopsy may have been a reflection of this tubular lesion. The increased creatinine and decreased chloride levels in the 125 mg/kg/day dose males could be a result of renal dysfunction.

Histopathological examination of the liver showed centrilobular hepatocyte hypertrophy to be present in males and females. This lesion without any evidence of cellular damage is characteristic of enzyme induction, and an adaptive response on the part of the liver to metabolism of the compound. This is likely to be reversible after cessation of treatment. It .is likely that this change would account for the increased liver weights in some groups receiving Ferrocene

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
30 mg/kg bw/day
Study duration:
chronic
Species:
dog
Quality of whole database:
Two good quality reliable studies are available.
In a peer reviewed chronic oral study, Hemosiderosis was observed in dogs dosed with ferrocene for 180 days at 30, 100, and 300 mg/kg. There was a dose related accumulation of iron. There was a reversible decrease in hemoglobin, packed cell volume, and erythrocyte counts with greatest change occurring during the first 28 days at doses of 300 mg/kg. Cirrhosis, and testicular hypoplasia were also observed at higher dose groups. Dogs observed for 12 to 26 months after the 180day treatment period showed no latent effects from massive iron overload. All other parameters were normal (Yeary 1969).

In another study, dosing Sprague-Dawley rats for 28d with Ferrocene via the diet produced effects on male and female RBC parameters leading to haemosiderin deposition in liver and spleen. In addition intracytoplasmic hyaline droplets in the kidney cortical tubular epithelium where seen. Depressions of body weight gain and food consumption were seen at 125 mg/kg/day dose level of both sexes (Innospec 1988)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Guidelines data not available. A study was made of the toxic effects in mice and rats of a 13 wk inhalation exposure to ferrocene according to methods published by Harkema et al., 1982
GLP compliance:
not specified
Species:
other: rat and mice
Strain:
other: F344/N rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
animals from Simonsen labs Inc.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
F344/N rats and B6C3F1 mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m3
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hr/day, 5 days/week
Remarks:
Doses / Concentrations:
3.0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
30 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Observations of rats were made after 5 days, 23 days and 13 weeks of exposure. Mice were not evaluated
Other examinations:
The respiratory function of male rats from each of the three exposed groups and one control group was measured after 30, 60, and 90 days of exposure according to previously published methods (Harkema et al.. 1982). These measurements included multiple assays of breathing patterns, lung volumes, mechanical properties oflung tissue, gas distribution uniformity, air flow limitation, and alveolar-capillary gas exchange efficiency. The tests were nondestructive, requiring only halothane anesthesia, and thus were performed serially on the same rats at each time.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Details on results:
Although histopathological lesions were observed in the nose, larynx, trachea, lung and liver of rats and mice and in kidneys of mice, the most severely affected tissue in both species was the nose. The severity of lesions was probably related to the high retention of ferrocene-introduced iron in the nose (Dahl and Briner, 1980) resulting from the high metabolic capacity of this tissue for ferrocene as a substrate (Sun et ai., 1991). Ferrocene is metabolized to hydroxyferrocene (Hanzlik et ai., 1978) which would decompose readily to release ferrous ion intracellularly. The ferrous ion could catalyze lipid peroxidation (Dumelin and Tappel, 1977) or hydroxyl-free radical formation by the Fenton reaction (Hewitt et aI., 1991). Either products of lipid peroxidation or the resulting free radicals could react with key cell components to account for the lesions observed.

The fact that the nasal olfactory mucosa was more affected than the nasal respiratory mucosa is probably due to the higher metabolic activity in the olfactory mucosa, which has been demonstrated in rats for ferrocene (Sun et aI., 1991). Other factors that might have contributed to the greater sensitivity of the olfactory compared to the nasal respiratory mucosa may include a higher tissue dose and a lower rate of chemical clearance. The nasal respiratory epithelium of mice was more affected by ferrocene exposure than that of rats. The metabolic capacity for ferrocene of the olfactory versus respiratory mucosa has not been examined in mice, nor has an interspecies comparison been made. Nasal olfactory versus nasal respiratory deposition of ferrocene has not been determined in either species. However, it has been shown that the mouse has a greater surface area of respiratory epithelium available for filtering air per unit volume of the nasal cavity than the rat (Gross el ai., 1982). This relative increase in area at risk might account for the observed differences in nasal respiratory lesions Nasal epithelial lesions have been classified according to whether they are induced by direct- or indirect-acting chemicals (Gaskell, 1990). Indirect-acting chemicals are metabolized to a toxic intermediate by the mixed-function oxidases known to be present in the olfactory mucosa (Hadley and Dahl, 1983; Dahl and Hadley, 1991). Direct-acting chemicals are those where the parent compound is toxic. The damage caused by ferrocene is similar to that caused by other indirect-acting chemicals, such as 3-trifluoromethylpyridine and 3-methylindole, in that all or a large percentage of the olfactory mucosa was affected, while the respiratory epithelium was relatively spared. Direct-acting chemicals, in contrast, usually cause injury with an anterior-posterior gradient of damage within the nose. The respiratory epithelium is damaged, while olfactory lesions, if they occur, are often restricted to the dorsal meatus (Gaskell, 1990).

The histopathologic findings in the 13-week study differ from those in our earlier 2-week study (Sun el a/., 1991) in that tissues and organs other than the nose accumulated inhaled ferrocene, as suggested by the histochemical stains for iron. Histopathologic lesions in these other tissues and organs were limited to pigment accumulation with degeneration of individual cells. This may reflect a lower capacity for ferrocene metabolism relative to olfactory tissue (known to be the case for rat liver (Sun et al, 1991), low penetration of the ferrocene vapor into those regions (Dahl and Briner, 1980), or the inability to detect subtle membrane damage using light microscopy. Furthermore, the cells in these other sites may have adequate intracellular mechanisms to protect against lipid peroxidation induced by low levels of intracellular iron. Higher levels of intracellular iron may be required to overwhelm these protective mechanisms and to produce lesions observable by light microscopy. The fact that pigment accumulation with concomitant degeneration was observed in individual cells of the larynx, trachea, and bronchioles of both species, and the tit liver of mice, suggests that these cells can hydroxylate ferrocene to some degree, and that cell injury will occur in the cells that accumulate higher levels of ferrocene-derived iron.
The amount of ferrocene swallowed during or after exposure, including that ingested after mucociliary clearance and after fur licking, was not measured..

In summary, several toxicological responses were observed in F344/N rats and B6C3F1 mice after a 13-week inhalation exposure. to ferrocene vapor at nominal concentrations of 3.0, 10, and 30 mg/m3. These included exposure- related but minimal changes in body weights and organ weights and histopathological lesions in the larynx, trachea, lungs, liver, (kidneys only in mice), and most notably in the nasal epithelium. The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver). In the studies reported here, as in our earlier 2-week study (Sun et al., 1991), the nasal lesions were present in both sexes of rats and mice at all exposure concentrations. It is important to note that the two lowest concentrations that resulted in these lesions were at and below the current TL V for ferrocene (10 mg/m3). Only a chronic study could determine the potential carcinogenicity of inhaled ferrocene vapor.
Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: Liver weight
Critical effects observed:
not specified
Conclusions:
In a peer reviewed study report, no clinical signs were seen in any rats exposed to any dose. The mean iron lung burden in rats exposed to 30 mg/m3 for 90 days was 4 times greater than the burden in control rats. The relative liver weight of rats showed a dose-related increase, which was significant (p<0.05) at the highest dose level for the males and at the 2 highest dose levels for the females. The relative liver weight of female mice was decreased at 3.0 mg/m3, but there was no dose-response relationship. On the other hand, the absolute liver weight of female mice showed a dose-related decrease, which was significant at all dose levels (p<0.05). The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver).
No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed. There w ere neither indications of developing pulmonary fibrosis nor of any haematological toxicity. Exposure-related histological alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium, where pigment accumulation, necrotising inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity which was dependent on the exposure concentration. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA
The material can be considered to have an LOAEL of 3 mg/m3 air
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3 mg/m³
Study duration:
subchronic
Species:
other: rat and mouse
Quality of whole database:
Two good quality peer reviewed studies available.

In a peer reviewed 90d inhalation study report, no clinical signs were seen in any rats exposed to any dose (0, 3.0, 10, and 30 mg ferrocene vapor/m3).

However liver weight changes were seen to all rats at all dose levels. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA.

The material can be considered to have an LOAEC of 3 mg/m3 air (Nikula KJ et al, 1993).

In a peer reviewed study to determine the toxic effects in mice and rats of a 2 wk inhalation exposure to ferrocene and of its in-vitro metabolism by rat nasal tissue and liver microsomes a NOAEC was found to be 5.0mg/m3 (Sun et al, 1991)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Guidelines data not available. A study was made of the toxic effects in mice and rats of a 13 wk inhalation exposure to ferrocene according to methods published by Harkema et al., 1982
GLP compliance:
not specified
Species:
other: rat and mice
Strain:
other: F344/N rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
animals from Simonsen labs Inc.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
F344/N rats and B6C3F1 mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m3
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hr/day, 5 days/week
Remarks:
Doses / Concentrations:
3.0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
30 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Observations of rats were made after 5 days, 23 days and 13 weeks of exposure. Mice were not evaluated
Other examinations:
The respiratory function of male rats from each of the three exposed groups and one control group was measured after 30, 60, and 90 days of exposure according to previously published methods (Harkema et al.. 1982). These measurements included multiple assays of breathing patterns, lung volumes, mechanical properties oflung tissue, gas distribution uniformity, air flow limitation, and alveolar-capillary gas exchange efficiency. The tests were nondestructive, requiring only halothane anesthesia, and thus were performed serially on the same rats at each time.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Details on results:
Although histopathological lesions were observed in the nose, larynx, trachea, lung and liver of rats and mice and in kidneys of mice, the most severely affected tissue in both species was the nose. The severity of lesions was probably related to the high retention of ferrocene-introduced iron in the nose (Dahl and Briner, 1980) resulting from the high metabolic capacity of this tissue for ferrocene as a substrate (Sun et ai., 1991). Ferrocene is metabolized to hydroxyferrocene (Hanzlik et ai., 1978) which would decompose readily to release ferrous ion intracellularly. The ferrous ion could catalyze lipid peroxidation (Dumelin and Tappel, 1977) or hydroxyl-free radical formation by the Fenton reaction (Hewitt et aI., 1991). Either products of lipid peroxidation or the resulting free radicals could react with key cell components to account for the lesions observed.

The fact that the nasal olfactory mucosa was more affected than the nasal respiratory mucosa is probably due to the higher metabolic activity in the olfactory mucosa, which has been demonstrated in rats for ferrocene (Sun et aI., 1991). Other factors that might have contributed to the greater sensitivity of the olfactory compared to the nasal respiratory mucosa may include a higher tissue dose and a lower rate of chemical clearance. The nasal respiratory epithelium of mice was more affected by ferrocene exposure than that of rats. The metabolic capacity for ferrocene of the olfactory versus respiratory mucosa has not been examined in mice, nor has an interspecies comparison been made. Nasal olfactory versus nasal respiratory deposition of ferrocene has not been determined in either species. However, it has been shown that the mouse has a greater surface area of respiratory epithelium available for filtering air per unit volume of the nasal cavity than the rat (Gross el ai., 1982). This relative increase in area at risk might account for the observed differences in nasal respiratory lesions Nasal epithelial lesions have been classified according to whether they are induced by direct- or indirect-acting chemicals (Gaskell, 1990). Indirect-acting chemicals are metabolized to a toxic intermediate by the mixed-function oxidases known to be present in the olfactory mucosa (Hadley and Dahl, 1983; Dahl and Hadley, 1991). Direct-acting chemicals are those where the parent compound is toxic. The damage caused by ferrocene is similar to that caused by other indirect-acting chemicals, such as 3-trifluoromethylpyridine and 3-methylindole, in that all or a large percentage of the olfactory mucosa was affected, while the respiratory epithelium was relatively spared. Direct-acting chemicals, in contrast, usually cause injury with an anterior-posterior gradient of damage within the nose. The respiratory epithelium is damaged, while olfactory lesions, if they occur, are often restricted to the dorsal meatus (Gaskell, 1990).

The histopathologic findings in the 13-week study differ from those in our earlier 2-week study (Sun el a/., 1991) in that tissues and organs other than the nose accumulated inhaled ferrocene, as suggested by the histochemical stains for iron. Histopathologic lesions in these other tissues and organs were limited to pigment accumulation with degeneration of individual cells. This may reflect a lower capacity for ferrocene metabolism relative to olfactory tissue (known to be the case for rat liver (Sun et al, 1991), low penetration of the ferrocene vapor into those regions (Dahl and Briner, 1980), or the inability to detect subtle membrane damage using light microscopy. Furthermore, the cells in these other sites may have adequate intracellular mechanisms to protect against lipid peroxidation induced by low levels of intracellular iron. Higher levels of intracellular iron may be required to overwhelm these protective mechanisms and to produce lesions observable by light microscopy. The fact that pigment accumulation with concomitant degeneration was observed in individual cells of the larynx, trachea, and bronchioles of both species, and the tit liver of mice, suggests that these cells can hydroxylate ferrocene to some degree, and that cell injury will occur in the cells that accumulate higher levels of ferrocene-derived iron.
The amount of ferrocene swallowed during or after exposure, including that ingested after mucociliary clearance and after fur licking, was not measured..

In summary, several toxicological responses were observed in F344/N rats and B6C3F1 mice after a 13-week inhalation exposure. to ferrocene vapor at nominal concentrations of 3.0, 10, and 30 mg/m3. These included exposure- related but minimal changes in body weights and organ weights and histopathological lesions in the larynx, trachea, lungs, liver, (kidneys only in mice), and most notably in the nasal epithelium. The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver). In the studies reported here, as in our earlier 2-week study (Sun et al., 1991), the nasal lesions were present in both sexes of rats and mice at all exposure concentrations. It is important to note that the two lowest concentrations that resulted in these lesions were at and below the current TL V for ferrocene (10 mg/m3). Only a chronic study could determine the potential carcinogenicity of inhaled ferrocene vapor.
Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: Liver weight
Critical effects observed:
not specified
Conclusions:
In a peer reviewed study report, no clinical signs were seen in any rats exposed to any dose. The mean iron lung burden in rats exposed to 30 mg/m3 for 90 days was 4 times greater than the burden in control rats. The relative liver weight of rats showed a dose-related increase, which was significant (p<0.05) at the highest dose level for the males and at the 2 highest dose levels for the females. The relative liver weight of female mice was decreased at 3.0 mg/m3, but there was no dose-response relationship. On the other hand, the absolute liver weight of female mice showed a dose-related decrease, which was significant at all dose levels (p<0.05). The effects of ferrocene exposures on organ weights may indicate a secondary response to the loss of appetite from the severe nasal lesions or may indicate that these are target organs for chronic toxicity (particularly the liver).
No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed. There w ere neither indications of developing pulmonary fibrosis nor of any haematological toxicity. Exposure-related histological alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium, where pigment accumulation, necrotising inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity which was dependent on the exposure concentration. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA
The material can be considered to have an LOAEL of 3 mg/m3 air
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3 mg/m³
Study duration:
subchronic
Species:
other: rat and mouse
Quality of whole database:
Two good quality peer reviewed studies available.

In a peer reviewed 90d inhalation study report, no clinical signs were seen in any rats exposed to any dose (0, 3.0, 10, and 30 mg ferrocene vapor/m3).

However liver weight changes were seen to all rats at all dose levels. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA.

The material can be considered to have an LOAEC of 3 mg/m3 air (Nikula KJ et al, 1993).

In a peer reviewed study to determine the toxic effects in mice and rats of a 2 wk inhalation exposure to ferrocene and of its in-vitro metabolism by rat nasal tissue and liver microsomes a NOAEC was found to be 5.0mg/m3 (Sun et al, 1991)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two good quality reliable studies are available for repeated dose oral toxicity.

In a peer reviewed chronic oral study of ferrocene, hemosiderosis was observed in dogs dosed with ferrocene for 180days at 30, 100, and 300 mg/kg. There was a dose related accumulation of iron. There was a reversible decrease in hemoglobin, packed cell volume, and erythrocyte counts with greatest change occurring during the first 28days at doses of 300 mg/kg. Cirrhosis, and testicular hypoplasia were also observed at higher dose groups. Dogs observed for 12 to 26 months after the 6 month treatment period showed no latent effects from massive iron overload. All other parameters were normal (Yeary 1969).

Orally dosing Sprague-Dawley rats for 28d with Ferrocene via the diet, produced effects on male and female RBC parameters leading to haemosiderin deposition in liver and spleen. In addition intracytoplasmic hyaline droplets in the kidney cortical tubular epithelium where seen. Depressions of body weight gain and food consumption were seen at 125 mg/kg/day dose level of both sexes (Innospec 1988).

Two good quality peer reviewed studies available for repeated dose inhalation toxicity.

In a 90d inhalation study report, no clinical signs were seen in any rats exposed to any dose (0, 3.0, 10, and 30 mg ferrocene vapor/m3). However liver weight changes were seen to all rats at all dose levels. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen clinical chemistry, and haematological parameters were observed The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalysed lipid peroxidation of cellular membranes or the iron-catalysed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA. The material can be considered to have an LOAEC of 3 mg/m3 air (Nikula KJ et al, 1993).

In a peer reviewed study to determine the toxic effects in mice and rats of a 14day inhalation exposure to ferrocene and of its in-vitro metabolism by rat nasal tissue and liver microsomes a NOAEC was found to be 5.0mg/m3 (Sun et al, 1991).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The 180d (chronic) study achieved a LOAEL

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A 90d inhalation study obtained the lowest LOAEC

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
A 90d inhalation study obtained the lowest LOAEC

Justification for classification or non-classification

According to the EU Classification, Labelling and Packaging Directive, Ferrocene should be classified for repeated exposure toxicity as adverse findings were found in the studies

Ferrocene should be classified as Specific Target Organ toxicity-repeated exposure Category 2 for Oral and Inhalation exposure (CLP) and as R48/20/22 (DPD)